Fluorescence-Based Activity Screening Assay Reveals Small Molecule Inhibitors of Vaccinia Virus mRNA Decapping Enzyme D9

Vaccinia virus (VACV) represents a family of poxviruses, which possess their own decapping machinery as a part of their strategy to eliminate host mRNAs and evade the innate immune response. D9 is one of the two encoded VACV decapping enzymes that is responsible for cap removal from the 5′ end of both host mRNA transcripts and viral double-stranded RNAs. Little is known about the structural requirements for D9 inhibition by small molecules. Here, we identified a minimal D9 substrate and used it to develop a real-time fluorescence assay for inhibitor discovery and characterization. We screened a panel of nucleotide-derived substrate analogues and pharmacologically active candidates to identify several compounds with nano- and low micromolar IC50 values. m7GpppCH2p was the most potent nucleotide inhibitor (IC50 ∼ 0.08 μM), and seliciclib and CP-100356 were the most potent drug-like compounds (IC50 0.57 and 2.7 μM, respectively). The hits identified through screening inhibited D9-catalyzed decapping of 26 nt RNA substrates but were not active toward VACV D10 or human decapping enzyme, Dcp1/2. The inhibition mode for one of the compounds (CP-100356) was elucidated based on the X-ray cocrystal structure, opening the possibility for structure-based design of novel D9 inhibitors and binding probes.


■ INTRODUCTION
Eukaryotic mRNAs are co-transcriptionally modified by the addition of a 7-methylguanosine triphosphate moiety at the 5′ end. This modification, referred to as the 5′ cap, protects mRNA from premature degradation by 5′-exonucleases and is necessary for canonical translation initiation. Only specialized decapping enzymes can remove the 7-methylguanosine cap from an mRNA body, thereby exposing it to 5′-to-3′ degradation. Hence, mRNA decapping is an important process, aiding the control of gene expression. Eukaryotic cells produce several decapping enzymes that participate in bulk mRNA decay, mRNA quality control, and specialized degradation pathways. 1−4 Two major decapping enzymes involved in bulk mRNA degradation are Nudix hydrolase, Dcp2, which is involved in the 5′−3′ degradation pathway and scavenger decapping enzyme, DcpS, which cleaves m 7 G-capped structures released as the products of 3′−5′ mRNA decay. 5,6 Moreover, other proteins possessing intrinsic decapping activity have been identified and include Nudix family hydrolases (e.g., Nudt3 and Nudt16) and DXO decapping exoribonuclease protein, which is responsible for degrading aberrantly capped RNAs. 7 Precise control of the activity of those enzymes is crucial to maintain the cell homeostasis.
Some viruses express their own decapping machinery as a part of their strategy to inhibit the cellular translation and promote the synthesis of viral proteins. Till date, four virusencoded decapping enzymes have been described: D9 and D10 from vaccinia virus (VACV), g5R from African swine fever virus, and L375 Nudix enzyme from mimivirus. 8−11 All three viruses belong to the family of nucleocytoplasmic large DNA viruses.
The viral decapping enzymes differ greatly in the substrate specificity but share the catalytic motif and the ability to cleave m 7 GpppN-RNAs to release m 7 GDP as a product ( Figure 1A). They represent a class of phosphohydrolases that cleave nucleoside diphosphates linked to another moiety X (Nudix). This Nudix motif consists of a 23 amino acid characteristic sequence GX 5 EX 5 [UA]XREX 2 EEXGU, wherein U represents an aliphatic, hydrophobic residue, and X represents any amino acid. 12 Albeit VACV is not pathogenic to humans, it represents a family of large DNA viruses, including zoonotic viruses, which can jump to humans and/or are pathogenic to livestock and humans. D9 and D10 from VACV are therefore prototype viral decapping enzymes, the studies of which may aid in elucidating how viruses use their decapping machinery to avoid the innate immune response and influence the host translation.
To ensure mRNA stability and translational activity, VACV adds 5′ caps onto viral mRNAs using the virus-encoded capping machinery. 13−15 Early VACV mRNAs are modified to form either m 7 GpppA m or m 7 GpppG m caps, whereas intermediate and late transcripts carry only m 7 GpppA m caps. 16,17 D9 is expressed in the early stage of infection and 8 together with D10, it prevents accumulation of dsRNAs, thereby minimizing cellular antiviral response mediated by PKR and OAS/RNase L. 13,18 D9 exhibits specificity for N7methylated cap structures and a preference for longer RNA substrates (>30 nt). 8 Unmethylated (Gppp-capped) RNAs are not cleaved. The enzyme is strongly inhibited by uncapped RNA, whereas N7-methylated guanine nucleotides such as m 7 GpppG, m 7 GTP, and m 7 GDP are less potent D9 inhibitors. Both D9 and D10 enzymes trigger the degradation of multiple cellular and viral mRNAs to promote the selective translation of viral mRNAs 19 and reduce the levels of dsRNAs, which are the main trigger of innate immune response ( Figure 1B). Interestingly, VACV deficient in either D9 or D10 is more immunogenic due to dsRNA accumulation, replicates more efficiently in cancerous than in healthy cells, and thus acts as effective oncolytic virus. 20 Significant effort has been made to understand the role of D9 and D10 in VACV replication; nonetheless, the differential functions of these enzymes, their regulation, and interdependence remain elusive.
Structural and functional studies on cellular decapping enzymes, both in vitro and in vivo, can be greatly facilitated by the design of specific small-molecule ligands and inhibitors. 21−23 As such, in this work, we aimed to design molecular tools that could find utility in studying the activity of recombinant D9 and aid discovery of small-molecule inhibitors of this enzyme. Little has been determined about cap-related substrate specificity of D9 or design of specific inhibitors. The so-far-reported inhibition studies relied on isotopically labeled RNA substrates, the use of which entails relatively high cost and low throughput. Here, we sought to design a new simple approach that can be used to study the activity of VACV D9 and discover inhibitors via high-throughput screening (HTS). To this end, we developed fluorescence intensity-based (FLINT) assay with a small-molecule fluorescent probe ( Figure 2A). The designed method was adopted to the HTS format to screen an in-house library of nucleotide-derived compounds and a commercially available library of pharmacologically active compounds (LOPAC 1280 , Sigma Aldrich). The identified hits were verified on short RNA substrates and crossexamined against VACV D10 and human decapping enzyme, Dcp1-Dcp2. Finally, one of the most potent inhibitors was crystalized in complex with D9, providing a deeper insight into the inhibition mode.

■ RESULTS
Development of Activity-Based Assay for D9 Enzyme. We first synthesized a set of fluorescently labeled nucleotides as potential fluorogenic substrates for D9. Knowing that D9 specifically cleaves 7-methylguanosine-capped transcripts, all probes were equipped with a 7-methylguanine nucleotide moiety as a recognition element and a fluorescent tag sensitive to m 7 G presence as a cleavage-responsive element (Figure 2A). 7-Methylguanosine has been shown to act as a quencher of electron-rich fluorescent dyes, giving the basis for the development of fluorescent turn-on probes. 24 To select the most suitable probe in terms of substrate and fluorescent properties, we tested several fluorescent dyes [pyrene (Py), perylene (Pe), and boron dipyrromethene (BODIPY-FL)] and different oligophosphate chain lengths (from tri-to pentaphosphate; Figure 2B). The compounds were subjected to enzymatic cleavage by D9, and emission spectra were recorded over time ( Figure 2C,D). In all cases, the gradual increase in fluorescence intensity was observed, consistent with cleavage by D9 enzyme. The greatest changes were observed for probes labeled with pyrene, but the efficiency varied depending on the length of the oligophosphate chain. The system response decreased with the length of the oligophosphate (over 14-, 4-, and 3-fold enhancement of the fluorescence signal for tri-, tetra-, and pentaphosphate, respectively); triphosphates were also the most rapidly cleaved substrates ( Figure S1). Therefore, the pyrene-labeled m 7 GTP analogue (1b) was selected for further studies as a turn-on probe with the optimal substrate and emission properties. As m 7 GTP-Py is structurally different from natural D9 RNA substrates, we tested whether the hydrolysis mechanism remains similar. The probe was subjected to D9 cleavage, and the reaction progress was monitored using RP-HPLC ( Figure S2A,B). Appropriate fractions containing hydrolysis products were collected and identified by mass spectrometry ( Figure S2C−F), proving conclusively that D9 enzyme cleaves the probe liberating m 7 GDP and monophosphate of the rest of the molecule, that is, with the same regioselectivity as observed for capped RNA.
Probe 1b was then characterized in more detail and employed to develop high-throughput screening assay in 96well plates ( Figure 3A). Buffer compositions, concentrations of divalent metal ions, and protein-stabilizing agents were adjusted to optimize the reaction conditions. Manganese (Mn 2+ ) ions were essential for efficient enzyme activity ( Figure  S3A), whereas addition of BSA as a stabilizing protein significantly improved reproducibility ( Figure S3B). Initial hydrolysis rates for varying 1b concentrations were next determined by measuring fluorescence intensity changes as a function of time. Plotting the initial rates as a function of substrate concentrations enabled the determination of Michaelis−Menten kinetic parameters for the probe (K M ,  V max , and k cat ; Figure 3B). Based on those results, we established optimal conditions (Table 1) that ensured sufficient fluorescence signal response, proper reaction kinetics, high protein stability, and good reproducibility. Z′ factor 25 determined under the optimized conditions was 0.75 ( Figure  3C), indicating that the quality of the assay is sufficient for the application in high-throughput experiments.
Inhibition Studies. To identify the structural preferences for D9 inhibition by substrate analogues, the optimized D9 assay was employed to evaluate an "in-house" library consisting of unmodified mono-, di-, and trinucleotides and their analogues. To assess whether such approach is feasible, we first tested susceptibility of selected nucleotides to D9 cleavage. A set of 15 compounds with different modifications were incubated with D9 for 2 h under assay conditions. Most of the compounds were unsusceptible or did not undergo significant hydrolysis during initial 30 min of the analysis ( Figure S4).
Next, the half-maximal inhibitory concentrations (IC 50 ) were determined for all nucleotides from the library by observing the degradation of 1b in the presence of different concentrations of the putative inhibitor ( Figure 3D, Table S1). The determined IC 50 values varied between 0.077 and over 100 μM. The strongest D9 inhibitors (IC 50 below 1 μM, Figure  3E,F) were identified among mononucleotides containing m 7 G moiety and three or more phosphate groups, indicating that both positively charged 7-methylguanosine and negatively charged phosphate bridge are required for tight interactions with the protein. Dinucleotide cap analogues generally showed weaker inhibitory properties (IC 50 from 2.6 up to 46 μM) but relatively strong inhibition was observed for several dinucleoside 5′,5′-tetraphosphates, including m 7 G m pppCH 2 pG (IC 50 = 2.6 ± 0.4 μM) and m 7 Gp S ppp S m 7 G D3 (IC 50 = 3.6 ± 0.4 μM); the latter has been previously identified as an inhibitor of yeast decapping enzyme SpDcp1/Dcp2. 26 The most potent compound in the group of trinucleotides was m 7 G m pppG m pG with IC 50 value of ∼4 μM. Interestingly, both in the group of dinucleotides and trinucleotides, 2′-O methylation of 7methylguanosine notably increased inhibitory properties, suggesting that this modification might increase binding affinity for D9. At the same time, compounds that were 2′-O methylated either at 7-methylguanosine or within the second nucleotide showed the highest susceptibility to hydrolysis by D9 ( Figure S4), which might suggest that this is a binding affinity-related effect.
In addition, we screened a commercially available compound library consisting mostly drug-like compounds (LOPAC 1280 , Sigma-Aldrich). Compounds were screened at 30 μM under conditions given in Table 1 ( Figure 4A). The relative reaction progress for each compound was calculated by normalizing the initial rate of the reaction in the presence of the tested compound to the initial rate in the absence of any inhibitor. The criterion of the cutoff for inhibitor selection for further evaluation was set to 30% of the maximal initial rate (dotted line), which yielded six potential hits. The hits were then further evaluated, including determination of their IC 50 values ( Figure 4B,C). Three most potent compounds had IC 50 value below 10 μM, and they included an adenine derivative seliciclib (IC 50 = 0.57 ± 0.05 μM), quinazolinamine derivative CP-100356 (IC 50 = 2.7 ± 0.7 μM), and a purine derivative SCH 58261 (4.5 ± 0.8 μM) ( Figure 4D).
Verification of Inhibitory Properties toward D9 for the Identified Hits. To further validate the hits, we checked whether the three most potent inhibitors from the LOPAC library and the best hit from nucleotide-derived library inhibit D9-catalyzed decapping of short capped RNA transcripts. A 26-nt long RNA substrate co-transcriptionally capped with m 7 GpppApG (cap 0 structure) 27 was subjected to D9catalyzed hydrolysis in the presence of the evaluated compound at three different concentrations (1, 10, and 100 μM). The reaction progress was analyzed electrophoretically in reference to the reaction performed in the absence of the inhibitor ( Figure 5A). The band intensities corresponding to capped and uncapped RNAs were quantified densitometrically, and the decay of the substrate was plotted as a function of time ( Figures 5B,C, S5). All compounds showed inhibitory activity toward RNA decapping, and their relative potencies were in good agreement with IC 50 values determined by the fluorescence assay using probe 1b. Finally, we tested the specificity of the compounds in the context of VACV D10 and human decapping enzyme Dcp1/2, which are also m 7 GpppRNA hydrolases belonging to the Nudix family. We did not observe any inhibitory activity toward Dcp1/2 for the tested compounds at concentrations up to 100 μM, thereby confirming their selectivity toward D9 ( Figures 5D,E, S6). m 7 GpppCH 2 p and CP-100356 showed weak inhibitory activity toward D10 ( Figure S7). The most potent drug-like D9 inhibitor identified in this study, seliciclib, showed very good selectivity for D9.
Crystallographic Structure of Vaccinia Virus D9 with CP-100356. To better understand the mechanism of inhibition, we determined the crystal structure of wild-type D9 bound to inhibitor CP-100356 (P3_B7) to 1.78 Å (Figure  Table S1; (E) representative inhibition curves and (F) structures of the most potent compounds from mono-, di-, and trinucleotide groups. Data are shown as mean values from triplicate experiment ±SEM.  Table 3). This crystal form has two copies of D9 in the asymmetric unit (all-atom RMSD 1.811 Å), each bound to 1 molecule of CP-100356. The conformation of CP-100356bound D9 is nearly identical to a recently published structure of D9 bound to m 7 GDP product in the post-catalytic, inactive conformation (all-atom RMSD 2.55 Å). Our crystal structure reveals that the CP-100356 is positioned in the cap-binding pocket by continuous stacking of the 6,7-dimethoxyquinazoline moiety between aromatic residues F54 and Y158 in a manner similar to m 7 G cap recognition in the product-bound structure, PDB 7SEZ ( Figure 6B,C). These conserved residues are essential for efficient cap hydrolysis. 28 However, in the complex with CP-100356, Y158 is significantly reoriented compared to the position occupied in the complex with m 7 GDP ( Figure 6B,C), presumably to allow more efficient π−π stacking interaction with the aromatic quinazoline system  Table 2; (C) inhibition curves for identified inhibitorsone compound had to be tested separately due to its spectroscopic properties. Data are mean values ± SEM from three independent experiments; (D) structures of six most potent hits, and their IC 50 values are shown in Table 2. of the ligand. Unlike the m 7 G cap in PDB 7SEZ, which is oriented by hydrogen bond base pair mimicry between the guanine base and residues D151 and E16, the CP-100356 inhibitor is not oriented by hydrogen bonding. Rather, the 6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety extends into a small cavity past D151 and E16 ( Figure 6C). It is clear from our crystal structure that CP-100356 inhibits D9 cap hydrolysis by stabilizing the enzyme in an inactive conformation, preventing conserved aromatic residues F54 and Y158 from binding capped mRNA substrate. Attempts to crystallize D9 with the other most potent inhibitors identified from our screen, seliciclib (P4_A6) and SCH 58261 (P16_F10), were unsuccessful. Nonetheless, based on the similarity of these compounds to natural nucleobase moieties, we hypothesize that they likely bind in a similar manner, that is, by stacking between F54 and Y158.

■ CONCLUSIONS
Literature reports that VACV D9 requires RNA body for decapping activity, and that the decapping efficiency increases with the length of the RNA. 8 Surprisingly, in this work, we discovered a set of minimal substrates (activity probes) for D9 that consist of m 7 GTP molecule connected to a fluorescent tag via the terminal phosphate moiety. The activity probes were evaluated as D9 substrates to select the most efficient and responsive internally quenched probes suitable for highthroughput assay development. Pyrene-labeled m 7 GTP (probe 1b) had the most favorable properties, both in term of hydrolysis kinetics and fluorescent response upon cleavage. Probably both the m 7 G nucleotide and the pyrene moiety contribute to recognition by D9 because the corresponding unlabeled compounds such m 7 GTP and m 7 GpppG are very poor D9 substrates (as determined by RP HPLC). The probe was used to develop fluorescent HTS assay to screen for D9 inhibitors. The addition of Mn 2+ , beside Mg 2+ ions, and the presence of BSA were necessary to maintain high protein activity and stability. The optimized assay was characterized by a Z′ factor of 0.75, that is, suitable for HTS. The screening experiments were performed on two distinct compound libraries. The first library consisted of nucleotide derivatives, most of which were substrate analogues of varying sizes (mono-, di-, and trinucleotides), and the second was   ACS Chemical Biology pubs.acs.org/acschemicalbiology Articles commercially available LOPAC). The nucleotide screening revealed that highly negatively charged 7-methylguanosine mononucleotides are the most potent inhibitors of D9. The LOPAC screening revealed several heteroaromatic compounds as potential hits, which were then further evaluated to show good IC 50 values and verified inhibitory properties on a longer set of substrates. The most interesting hits identified in the screening were seliciclib and CP-100356. Seliciclib, a 2,6diaminopurine derivative, was the most potent D9 inhibitor, which combined with its very low inhibitory activity against D10 and hDcp1/2 making it a good candidate for future development. Albeit we were not able to determine the crystal structure of D9 in complex with this inhibitor, its structure suggests that the recognition may be mediated by amino acidforming nucleic acid-binding sites. CP-100356 was another potent and selective inhibitor of D9 with high potential of future development. The crystal structure of recombinant D9 in complex with CP-100356 was determined, which revealed that the compound targets the m 7 GDP-binding site and forms stacking interactions similar to those observed for 7methylguanine, suggesting a competitive inhibition mode. This, combined with the observed selectivity of the compound upon cross-examination with VACV D10 and human PNRC2-Dcp1/Dcp2 complex, indicates that the cap-binding site in D9 is distinctive enough from other decapping Nudix family hydrolases to enable the development of small molecules selectively targeting this chemical space.

■ METHODS
Expression and Purification of Recombinant Viral D9 and D10 Proteins. Recombinant viral D9 decapping enzyme with additional C-terminal His-tag sequence was expressed, as previously described. 28 The concentration of the protein was determined spectrophotometrically by assuming ε 280 = 14,900 M −1 cm −1 . The enzyme was stored at −80°C in a storage buffer (10 mM MES, pH 7.0, 300 mM NaCl, 1 mM DTT, and 10% glycerol). Viral D10 decapping enzyme was expressed following a similar protocol, and the detailed procedure will be published elsewhere.
Expression and Purification of Recombinant Human PNRC2-Dcp1/2 Complex. Human PNRC2-Dcp1/2 decapping complex genes were obtained in the plasmid vector (pET-Duet_PNRC2-Dcp1/2). Protein complex (for sequence details, see Figure S8) with His-tag was overexpressed in the E. coli BL21(DE3) RIL strain in LB medium with ampicillin (100 μg/mL). Cells were grown to OD 600 ∼0.5 at 37°C, then temperature was adjusted to 20°C . When the culture reached OD 600 of 0.6−0.7, protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to the final concentration of 1 mM, followed by overnight incubation at room temperature with shaking. Cells were then harvested by centrifugation (7000g, 4°C) and lysed by sonication in 50 mM sodium phosphate (pH 7.5), 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM DTT with lysozyme (1 mg/mL), and protease inhibitors (1 mM PMSF, 10.5 μM leupeptin, and 1 μM pepstatin A). The lysate was centrifuged at 40,000g for 30 min at 4°C , and the protein was purified by immobilized metal affinity chromatography on the GE Healthcare HisTrap HP column. The column was washed with 50 mM phosphate (pH 7.5), 1 M NaCl, 30 mM imidazole, and 1 mM DTT, and the PNRC2-Dcp1/2 complex was eluted using 50 mM phosphate buffer (pH 7.5), 300 mM NaCl, 500 mM imidazole, and 1 mM DTT. The supernatant was loaded onto the GE Healthcare HisTrap Heparin HP column and eluted with 50 mM sodium phosphate (pH 7.5), 1 M NaCl, and 1 mM DTT. The last step of purification was size exclusion chromatography on the GE Healthcare HiLoad 16/600 200 pg column. The purified human PNRC2-Dcp1/2 complex was concentrated using 30 kDa Amicon filters and flash frozen in liquid nitrogen. The concentration of the protein was determined spectrophotometrically by assuming ε 280 = 82,445 M −1 cm −1 . The enzyme was stored at −80°C in a storage buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT, and 20% glycerol).
Fluorescent Probe Synthesis. All solvents and chemical reagents for non-fluorescent nucleotide synthesis were purchased from Sigma-Aldrich and used without any pre-treatment, unless otherwise indicated. BODIPY FL iodoacetamide was acquired from ThermoFisher Scientific. Pyrene azide and perylene azide were purchased from Lumiprobe. Guanosine and guanosine 5′-monophosphate disodium salts were purchased from Carbosynth. m 7 GTPγS triethylammonium salt was synthesized, as described previously. 29 Probes 1b, 1d, and 1e were synthesized, as described previously. 24 Fluorescent nucleotides were purified using analytical or semipreparative HPLC. Analytical HPLC was performed on Agilent Tech. Series 1200 using the (RP)Supelcosil LC-18-T HPLC column (4.6 × 250 mm, flow rate 1.3 mL/min) with a linear gradient of acetonitrile (0−100% from 0 to 15 min) in 0.05 M ammonium acetate buffer (pH 5.9). UV detection was performed at 260 nm, as well as fluorescent label absorption and fluorescence maximum. Semi-preparative RP HPLC was performed on the same apparatus equipped with the Grace Vision HT C18 HL column (250 cm × 22 mm, 10 μm, flow rate 5.0 mL/min) with a linear gradient of acetonitrile (0−100% from 0 to 120 min) in 0.05 M ammonium acetate buffer (pH 5.9) and UV detection at 260 nm and at the fluorescent tag absorption and emission maximum. The structure and homogeneity of each final product were confirmed by RP HPLC and high-resolution mass spectrometry HRMS (ESI − ) with a Thermo Scientific LTQ Orbitrap Velos mass spectrometer. Spectroscopic Measurements. Fluorescence emission spectra were recorded using the Agilent Cary Eclipse fluorescence spectrophotometer equipped with the xenon lamp under temperature-controlled conditions. All experiments were performed at 37°C in the Hellma quartz cuvette (10 × 4 mm) with a sample volume 1000 μL in 10 mM Tris·HCl (pH 7.5), 100 mM KOAc, 2 mM MgCl 2 , 0.5 mM MnCl 2 , and 2 mM DTT. Before each measurement, the buffer was degassed. Emission spectra were registered for all excitation maxima (detailed wavelengths for each probe: 1aexc. 490/em. 512 nm; 1b, 1d, and 1e345/378 nm; and 1c420/489 nm).
Enzymatic reaction progress was examined after 5, 15, 30, 45, and 60 min. Aliquots were collected, and the reaction progress was terminated by heat inactivation for 3 min at 95°C, followed by centrifugation. The samples were analyzed by RP HPLC (Agilent Technologies Series 1200). Appropriate fractions from probe 1b hydrolysis were collected and identified based on the mass measurements. Mass spectra were recorded on the AB Sciex API 3200 spectrometer.
Point Fluorescence MeasurementsGeneral Information. Point fluorescence measurements were performed using the BioTek Synergy H1 microplate reader. Before each measurement, the reaction buffer was degassed. Experiments were performed in the Greiner 96well, black, non-binding plates at 30°C. Before the measurement, the plate was preincubated for 15 min at 30°C with mixing (300 rpm). The point fluorescence was registered with 1 min interval and detection at 345 nm excitation wavelength and 378 nm emission wavelength.
Kinetic Parameter Determination. To determine the kinetic parameters of enzymatic hydrolysis of probe 1b by D9 enzyme, point fluorescence measurements were performed. Each well of the plate contained an appropriate buffer (10 mM MOPS·HCl, pH 7.0, 100 mM KOAc, containing 2 mM MgCl 2 , 0.3 mM MnCl 2 , and 2 mM DTT) with addition of 0.1% BSA, a substrate (probe 1b at a concentration range 0−15 μM) and an enzyme (5 nM of D9) to a final volume of 150 μL. The measurement was carried out on until full saturation, which allowed us to convert the initial rates from au/min to mol/min/mg. The initial rates were calculated by fitting a linear curve to the first 10 points (10 min) of the hydrolysis reaction. To calculate the kinetic parameters, the Michaelis−Menten model was applied with the following formula fitted to the experimental data using GraphPad Prism software where V/V 0 is the relative initial rate of the reaction, I min and I max are bottom and top plateaus, and C inh is the concentration of the inhibitor. All tested compounds come from LOPAC 1280 ( Table 2) or our inhouse library of cap analogues (determined IC 50 values with compound's structures and synthesis references are available in Table S1).
RNA Synthesis. Short RNA sequences were synthesized by in vitro transcription (IVT) using T7 class II promoter Φ2.5 initiated by ATP (TAATACGACTCACTATTA). 32 The typical transcription reaction (200 μL) was carried out for 4 h at 37°C and contained 5 mM each of UTP/GTP/CTP (Thermo Fisher Scientific), 1.25 mM ATP (Thermo Fisher Scientific), and 6.5 mM of trinucleotide cap analogue (m 7 GpppApG), RNA Polymerase Buffer (Thermo Fisher Scientific), 1 μM of annealed oligonucleotides as DNA template (CAGTAATACGACTCACTATTAGGGAAGCGGGCATGCGGC-CAGCCATAGCCGATCA and TGATCGGCTATGGCTGGCCG-CATGCCCGCTTCCCTAATAGTGAGTCGTATTACTG), 1 U/ μL RiboLock RNase inhibitor (Thermo Fisher Scientific), 20 mM MgCl 2 , and T7 RNA polymerase (20 U/μL Thermo Fisher Scientific). Uncapped transcripts (IVT) were obtained as mentioned above but in the presence of 5 mM ATP and without addition of cap analogue. After 4 h incubation, 7 U of DNase I (Thermo Fisher Scientific) was added, and the mixture was incubated for another 30 min at 37°C. The reaction mixture was stopped by the addition of equimolar amounts of Na 2 EDTA water solution (EDTA to Mg 2+ ) and RNA phenol−chloroform extraction, followed by RNA precipitation (3 M NaOAc pH 5.2, ethanol). The crude RNA sample was next purified using the HPLC and Phenomenex Clarity 3 μM Oligo-RP column (150 × 4.6 mm) (the method is given below) and precipitated as before. To obtain a homogeneous RNAs at 3′ end, appropriate sample underwent trimming by DNAzyme 10−23 (TGATCGGCTAGGCTAGCTACAACGAGGCTGGCCGC). The reaction mixture contains equal molar concentrations of RNA substrates and DNAzyme in 50 mM Tris (pH 8.0) and 50 mM MgCl 2 for 1 h at 37°C. The reaction was then purified using HPLC, and the desired product was precipitated with ethanol as mentioned above. This procedure allows us to obtain a 3′ homogeneous 25-nt (uncapped) or 26-nt (capped) transcripts. The quality of the obtained short RNAs was checked on 15% acrylamide/7 M urea/TBE gels with SYBR Gold (Invitrogen) staining, and the concentration was determined spectrophotometrically.
RNA Decapping Assay. 20 ng of 26-nt capped RNA transcript was incubated with an appropriate decapping enzyme (3 nM D9 or 11 nM Dcp1/2 or 200 nM D10) in an appropriate buffer with addition of 0.1% BSA at 37°C. After being indicated, times reactions were terminated by adding equal volume of the loading dye (4.5 M urea, 50% formamide, 20 mM EDTA, 0.03% bromophenol blue, and 0.03% xylene cyanol) and flash freezing. Samples were then resolved by PAGE on denaturing 15% acrylamide/7 M urea/TBE gel and were stained with SYBR Gold (Invitrogen) and visualized using a Typhoon FLA 9500 (GE Healthcare). The band intensities corresponding to capped and uncapped RNAs were quantified densitometrically using 1D Gel Image Analysis Software TotalLab CLIQS.
X-ray Crystallography. The CP-100356 inhibitor was dissolved in DMSO at a concentration of 5 mg/mL. The protein−inhibitor complex was prepared by mixing D9 and CP-100356 in crystallization buffer containing 20 mM MgCl 2 to a final concentration of 10 mg/mL D9 and 3 mM cap analogue substrates and incubated at room temperature for 30 min. The protein−substrate complex was mixed with well solution at a 1:1 ratio, and crystals were grown by hanging drop vapor diffusion at room temperature. D9:CP-100356 well solution contained 20% PEG 3350 and 100 mM NaF. Crystals were flash frozen in liquid nitrogen using a cryoprotectant consisting of well solution with 25% glycerol. All data sets were collected on beamline 8.3.1 at the Advanced Light Source at 100 K and wavelength 1.11583 Å using the Pilatus3 S 6 M detector and indexed, integrated, and scaled using XDS, 33 Pointless and Aimless 34 via automated beamline software ELVES. 35 Phasing was carried out by molecular replacement with the previously solved D9 structure in the post-catalytic conformation, 7SEZ. 28 The structure was then iteratively refined in PHENIX 36 and manually adjusted in COOT. 37 ■ ASSOCIATED CONTENT
Detailed results of inhibitory studies obtained from FLINT assay, kinetic and hydrolysis experiments, HTS assay optimization, susceptibility to D9 hydrolysis, in vitro decapping assay with PAGE gels and sequences of the used proteins, and HPLC profiles and HRMS spectra for newly synthesized compounds (PDF)